Phenylboronic acid complexes for bioconjugate preparation

ABSTRACT

The present invention relates to a novel class of phenylboronic acid bioconjugate complexes derived from aminosalicylic acid, and the method of making and using such bioconjugate complexes. The bioconjugate complexes are in the form of the following general formulas; ##STR1## wherein group X is selected from the group consisting of O, NH, N-alkyl, NC 6  H 5 , N-aryl, NCH 2  -aryl, NCH 2  CH 2  OH, NCOCH 2  CH 2  OH, NOH, NO-alkyl and NOCH 2  -aryl, wherein alkyl denotes a hydrocarbon moiety of from 1 to 4 carbons in length and which may be linear or branched, wherein aryl is selected from the group consisting of an aromatic ring, a substituted aromatic ring and a fused aromatic ring, 
     wherein group Y is selected from the group consisting of O, S, NH, N-alkyl, N-aryl and NCH 2  -aryl, 
     wherein groups Z and Z* comprise a spacer selected from an alkyl and a polyethyleneglycol chain, of from 1 to 16 carbon equivalents in length, wherein the chain may contain intermediate amide and disulfide bonds, and 
     and wherein groups BAS and BAS* are bioactive species.

This is a divisional of application Ser. No. 08/188,958, filed Jan. 28, 19994 now U.S. Pat. No. 5,594,111.

FIELD OF THE INVENTION

The present invention relates to the field of bioconjugate preparation, and more particularly, to a class of phenylboronic acid complexs useful for the conjugation of biological macromolecules, and the method of making and using such complexes.

BACKGROUND OF THE INVENTION

Bioconjugation is a descriptive term for the joining of two or more different molecular species by chemical or biological means, in which at least one of the molecular species is a biological macromolecule. This includes conjugation of proteins, peptides, polysaccharides, lectins, hormones, nucleic acids, liposomes and cells, with each other or with any other molecular species that add useful properties, including radionuclides, toxins, haptens, inhibitors, fluorophores, ligands, etc. Immobilization of biological macromolecules is also considered a special case of bioconjugation in which the macromolecule is conjugated, either reversibly or irreversibly, to an insoluble support. Bioconjugation is utilized extensively in biochemical, immunochemical and molecular biological research. Applications of bioconjugation are numerous, and include affinity chromatography, affinity cytochemistry, histochemistry, pathological probe detection, diagnostics, signal amplification, immunoassay, hybridoma technology, blotting technology, bioaffinity sensors, gene probe detection, cross-linking reagents, affinity targeting, affinity perturbation, drug delivery, fusogenic reagents, immobilizing reagents, selective retrieval, selective elimination, flow cytometry and cytological probe detection.

Avidin-Biotin System

Although numerous methods of bioconjugate preparation have been described, a significant number of those reported in the literature have been prepared by exploiting the Avidin-Biotin system, in which, the binding specificity of the protein Avidin (purified from egg white), or Streptavidin (purified from the bacterium Streptomyces avidinii), toward the cofactor Biotin (vitamin H) is utilized to bridge an Avidin conjugated macromolecule with a biotinylated macromolecule. Both Avidin and Streptavidin possess four Biotin binding sites of very high affinity (K=10¹⁵ mol⁻¹). This system has been utilized extensively for enzyme-linked immuno solid-phase assay (ELISA), in which an enzyme-Avidin conjugate (useful for detection by reaction with the enzyme's substrate to afford a colored or chemiluminescent product) is employed to detect the presence of a biotinylated antibody, after first binding the antibody to an immobilized antigen or hapten. Applications of the Avidin-Biotin system number in the hundreds, and have recently been reviewed (Wilchek, M. and Bayer, E. A. (1990) Methods in Enzymology, 184). Although utilized extensively, several limitations are known to be associated with the Avidin-Biotin system, which include nonspecific binding generally attributed to the basicity of the Avidin molecule, nonspecific binding attributed to the presence of carbohydrate residues on the Avidin molecule, and background interference associated with the presence of endogenous Biotin, which is ubiquitous in both eukaryotic and prokaryotic cells.

Digoxigenin Anti-Digoxigenin System

An alternative bioconjugation system designed to overcome the limitations associated with the Avidin-Biotin system has recently been developed for the detection of gene probes by ELISA (Kessler, C., Holtke, H.-J., Seibl, R., Burg, J. and Muhlegger, K. (1990) Biol. Chem. Hoppe-Seyler, 371,917-965). This system involves the use of the steroid hapten Digoxigenin, an alkaloid occuring exclusively in Digitalis plants, and Fab fragments derived from polyclonal sheep antibodies against Digoxigenin (anti-Digoxigenin). The high specificity of the various anti-Digoxigenin antibodies affords low backgrounds and eliminates the non-specific binding observed in Avidin-Biotin systems. Digoxigenin-labeled DNA and RNA probes can detect single-copy sequences in human genomic Southern blots. The development of the Digoxigenin anti-Digoxigenin system has recently been reviewed (Kessler, C. (990) in Advances in Mutagenesis Research (Obe, G. ed.) pp. 105-152, Springer-Verlag, Berlin/Heidelberg).

Immobilized Boronates

Phenylboronic acids are known to interact with a wide range of polar molecules having the requisite functionalities. Complexes of varying stability, involving 1,2-diols, 1,3-diols, 1,2-hydroxy acids, 1,3-hydroxy acids, 1,2-hydroxylamines, 1,3-hydroxylamines, 1,2-diketones and 1,3-di-ketones, are known to form with either neutral phenylboronic acid or phenylboronate anion. Consequently, immobilized phenylboronic acids have been exploited as chromatographic media to selectively retain, from complex samples, those molecular species having the requisite functionalities. Many important biological molecules including carbohydrates, catecholamines, prostaglandins, ribonucleosides, and steroids contain the requisite functionalities, and have been either analyzed or purified in this manner. The use of immobilized phenylboronates for the isolation and separation of biological molecules has been reviewed (Singhal, R. P. and DeSilva, S. S. M. (1989) Adv. Chromatog., 4, 124-130; Mazzeo, J. R. and Krull. I. S., BioChromatog., 4, 124-130 (1989); and Bergold, A. and Scouten, W. H. (1983) in Solid Phase Biochemistry (Scouten, W. H. ed.) pp. 149-187, John Wiley & Sons, New York).

Phenylboronic acid, like boric acid, is a Lewis acid, and ionizes not by direct deprotonation, but by hydration to give the tetrahedral phenylboronate anion (pK_(a) =8.86). Phenylboronic acid is three times as strong an acid as boric acid. Ionization of phenylboronic acid is an important factor in complexation, in that, upon ionization, boron changes from trigonal coordination (having average bond angles of 120° and average bond lengths of 1.37 Å) to the tetrahedrally coordinated anion (having average bond angles of 109° and average bond lengths of 1.48 Å).

A number of phenylboronic acids have been described in the literature for use in preparation of immobilized phenylboronic acid chromatographic supports. Several of these compounds and their corresponding pK_(a) values are illustrated below. Contemporary research in this field is directed toward the development of phenylboronic acids with pK_(a) values below that of (3-aminophenyl)boronic acid (pK_(a) 8.75), which is presently utilized in the preparation of all commercial phenylboronic acid chromatographic supports. The development on an immobilized phenylboronic acid chromatographic support with a significantly lower pK_(a) then that of (3-aminophenyl)boronic acid would enable the retention of a variety of biomolecules under physiological conditions (pH 7.4), thereby substantially extending the breath of compounds suitable for analysis by the method. ##STR2##

Compounds having cis or coaxial 1,2-diol and 1,3-diol functionalities, and particularly carbohydrates, are presently known to complex with immobilized phenylboronate anion, to form cyclic esters, only under alkaline aqueous conditions (Lorand, J. P. and Edwards, J. O. (1959) J. Org. Chem. 24, 769).

Acidification of 1,2-diol and 1,3-diol complexes is known to release the diol containing species, presumably due to hydrolysis of the cyclic ester, which is induced by ring-strain associated with a five-membered cyclic boronic acid ester involving trigonally coordinated boron. Coplaner aromatic 1,3-diols, like 1,8-dihydroxynaphthalene, are known to complex even under acidic conditions due to the hydrolytic stability of six-membered cyclic boronic acid esters (Sienkiewicz, P. A. and Roberts, D. C. (1980), J. Inorg. Nucl. Chem., 42, 1559-1571).

Substituted phenols having pendant 1,3-hydroxylamide, 1,3-hydroxyamidine and 1,3-hydroxyoxime moieties are also known to complex reversibly with borate buffer, under alkaline aqueous conditions (Tanner, D. W. and Bruice, T. C. (1967) J. Amer. Chem. Soc., 89, 6954-6971), in a manner analogous to that in which phenylboronic acids are known to complex.

Although immobilized phenylboronates have been utilized for chromatographic separation of biological molecules having the requisite functionalities, notwithstanding the substantial amount of research into bioconjugation, and the substantial amount of investment in this field, the selectivity of phenylboronic acid has not heretofore been exploited to enable the conjugation of biological macromolecules with one another or with other molecular species that add useful properties.

SUMMARY OF THE INVENTION Definitions

As used herein the following terms have the following meanings:

Bioactive species refers to a compound preferably selected from, but not limited to, proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes, cells, drugs, radio-nuclides, toxins, haptens, inhibitors, fluorophores, ligands and solid-phase supports.

Phenylboronic acid complexing reagent refers to a reagent comprised of a phenylboronic acid complexing moiety and a reactive moiety suitable for appending a phenylboronic acid complexing moiety to a bioactive species (see FIGS. 1, 3 and 4, General Formula V). Phenylboronic acid complexing reagents are described in greater detail in my copending application "Phenylboronic Acid Complexing Reagents Derived From Aminosalicylic Acid", Ser. No. 08/188,531, now U.S. Pat. No. 5,594,151, which is incorporated herein by reference.

Phenylboronic acid complexing semiconjugate refers to a bioactive species having a pendant phenylboronic acid complexing moiety which is derived from the reaction of a bioactive species with a phenylboronic acid complexing reagent (see FIGS. 1, 3 and 4).

Phenylboronic acid complexing cross-linking reagent refers to a reagent comprised of two phenylboronic acid complexing moieties separated by a spacer (see FIG. 2, General Formula VI). Phenylboronic acid complexing cross-linking reagents are described in greater detail in my copending application "Phenylboronic Acid Complexing Reagents Derived From Aminosalicylic Acid", Ser. No. 08/188,531, now U.S. Pat. No. 5,594,151, which is incorporated herein by reference.

Phenylboronic acid reagent refers to a reagent comprised of a phenylboronic acid moiety and a reactive moiety suitable for appending a phenylboronic acid moiety to a bioactive species (see Figures I and II, General Formula VII).

Phenylboronic acid semiconjugate refers to a bioactive species having a pendant phenylboronic acid moiety which is derived from the reaction of a bioactive species with a phenylboronic acid reagent (see FIGS. 1 and 2).

Phenylboronic acid cross-linking reagent refers to a reagent comprised of two phenylboronic acid moieties separated by a spacer (see FIGS. 3 and 4, General Formulas VIII and IX, respectively).

Bioconjugate complex refers to the product formed upon reaction of a phenylboronic acid complexing semiconjugate with a phenylboronic acid semiconjugate.

The present invention relates to a novel class of bioconjugates derived from phenylboronic acid complexes, and the method of making and using such bioconjugate complexes. In the present invention, in the place of prior art Avidin-Biotin and Digoxigenin-anti-Digoxigenin systems, phenylboronic acid complexes are utilized to facilitate chemical conjugation of bioactive species without the use of intermediary biological macromolecules. Bioconjugate complexes suitable for conjugation of two or more bioactive species, for the purpose of preparing bioconjugates, are selected from either General Formulas I, II, III and IV, set forth below. ##STR3##

Bioconjugate complexes of General Formula I are those wherein group X is selected from either O, NH, N-alkyl, NC₆ H₅, N-aryl, NCH₂ -aryl, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH, NO-alkyl and NOCH₂ -aryl, wherein alkyl denotes an alkyl hydrocarbon moiety of from 1 to 4 carbons in length, wherein the chains may be branched, wherein aryl is selected from either an aromatic ring, a substituted aromatic ring and fused aromatic rings, wherein group Y is selected from either O, S, NH, N-alkyl, N-aryl and NCH₂ -aryl, wherein alkyl and aryl are as was previously defined, wherein groups Z and Z* comprise a spacer selected from either an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain, up to 16 carbon equivalents in length, wherein either chain may contain intermediate amide and disulfide bonds, and wherein groups BAS and BAS* are bioactive species.

Bioconjugate complexes of General Formula I are preferably those wherein group X is selected from, but not limited to, either O, NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH and NOCH₃, wherein group Y is preferably O, and wherein groups Z and Z* are preferably selected from, but not limited to, either (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to 4, and wherein BAS and BAS* are bioactive species. ##STR4##

Bioconjugate complexes of General Formula II are those wherein group X is selected from either O, NH, N-alkyl, NC₆ H₅, N-aryl, NCH₂ -aryl, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH, NO-alkyl and NOCH₂ -aryl, wherein alkyl denotes an alkyl hydrocarbon moiety of from 1 to 4 carbons in length, wherein the chains may be branched, wherein aryl is selected from either an aromatic ring, a substituted aromatic ring and fused aromatic rings, wherein group Y is selected from either O, S, NH, N-alkyl, N-aryl and NCH₂ -aryl, wherein alkyl and aryl are as was previously defined, wherein groups Z and Z* comprise a spacer selected from either an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain, up to 16 carbon equivalents in length, wherein either chain may contain intermediate amide and disulfide bonds, wherein group Z is appended to two phenylboronic acid complexing moieties, and wherein group BAS* is a bioactive species.

Bioconjugate complexes of General Formula II are preferably those wherein group X is selected from, but not limited to, either O, NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH and NOCH₃, wherein group Y is preferably O, and wherein groups Z and Z* are preferably selected from, but not limited to, either (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to 4, and wherein BAS* is a bioactive species. ##STR5##

Bioconjugate complexes of General Formulas III and IV are those wherein group X is selected from either O, NH, N-alkyl, NC₆ H₅, N-aryl, NCH₂ -aryl, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH, NO-alkyl and NOCH₂ -aryl, wherein alkyl denotes an alkyl hydrocarbon moiety of from 1 to 4 carbons in length, wherein the chains may be branched, wherein aryl is selected from either an aromatic ring, a substituted aromatic ring and fused aromatic rings, wherein group Y is selected from either O, S, NH, N-alkyl, N-aryl and NCH₂ -aryl, wherein alkyl and aryl are as was previously defined, wherein groups Z and Z* comprise a spacer selected from either an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain, up to 16 carbon equivalents in length, wherein either chain may contain intermediate amide and disulfide bonds, wherein group Z* is appended to two phenylboronic acid moieties, and wherein group BAS is a bioactive species.

Bioconjugate complexes of General Formula II are preferably those wherein group X is selected from, but not limited to, either O, NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH and NOCH₃, wherein group Y is preferably O, and wherein groups Z and Z* are preferably selected from, but not limited to, either (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to 4, and wherein BAS* is a bioactive species.

Bioconjugate complexes of General Formula I are employed to conjugate two different bioactive species, for example, to conjugate an enzyme with an antibody for use in an ELISA assay, to conjugate a nucleic acid probe with a fluorophore to facilitae detection of a genomic sequence, and to conjugate a toxin to a monoclonal antibody for use in targeted drug delivery. Bioconjugate complexes of General Formula I comprise the most general application of the method of bioconjugate preparation described herein.

Bioconjugate complexes of General Formulas II, III and IV are employed to conjugate identical bioactive species by cross-linking bioactive species having pendant phenylboronic acid complexing moities into macromolecular aggregates. Aggregates of this type involving enzymes are useful for increasing detection limits in ELISA and related assays by substantially increasing the effective concentration of enzyme available for conversion of colorless substrate into detectable product. Similarly, fluorophore labeled proteins having pendant phenylboronic acid complexing moieties my be aggregated in this manner to improve their visual or spectrophotometric detection. Aggregates having excess pendant phenylboronic acid moities may be further conjugated with other bioactive species having pendant phenylboronic acid complexing moieties (phenylboronic acid complexing semiconjugates). This general approach is analogous to the preparation of sandwich type assays involving the Avidin Biotin system.

Phenylboronic acid cross-linking reagents of General Formula VI (see FIG. 2) may be employed, by reaction in large excess, and subsequent removal of excess reagent, to convert bioactive species having pendant phenylboronic acid moities (phenylboronic acid semiconjugates) into bioactive species having pendant phenylboronic acid complexing moities (phenylboronic acid complexing semiconjugates).

Similarly, phenylboronic acid cross-linking reagents of General Formulas VIII and IX (see FIGS. 3 and 4) may be employed, by reaction in large excess, and subsequent removal of excess reagent, to convert bioactive species having pendant phenylboronic acid complexing moities (phenylboronic acid complexing semiconjugates) into bioactive species having pendant phenylboronic acid moities (phenylboronic acid semiconjugates).

Bioconjugate complexes of General Formulas I, II, III and IV are prepared in either buffered aqueous solutions, organic solvents and aqueous solutions containing organic solvents. The complex is formed within a few minutes at room temperature. Preparation of the bioconjugate complex is insensitive to significant variations in ionic strength, temperature, and the presence of chaotropic agents (protein denaturants), which are incompatible with prior art systems wherein the structure of a biological macromolecule must be maintained to preserve requisite binding properties. In most instances, the constraints governing the formation of bioconjugate complexes, by the system described herein, are limited to the selection of an appropriate pH and whatever additional limitations are imposed by the conditions required to maintain viability of the bioactive species.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the preparation of bioconjugate complexes of General Formula I.

FIG. 2 illustrates the preparation of bioconjugate complexes of General Formula II.

FIG. 3 illustrates the preparation of bioconjugate complexes of General Formula III.

FIG. 4 illustrates the preparation of bioconjugate complexes of General Formula IV.

FIG. 5 illustrates the synthetic preparation of reagents of General Formula VII.

FIG. 6 illustrates both the trigonal and tetrahedral forms of bioconjugate complexes of General Formulas I to IV.

FIG. 7 illustrates tautomeric and conformation isomers of semiconjugates derived from reagents of General Formulas I to IV.

FIG. 8. illustrates tautomeric and conformation isomers of bioconjugates complexes of General Formulas I to IV.

FIG. 9 illustrates the formation of bioconjugate complexes of General Formulas I to IV, wherein Q is O, and wherein R is O.

DETAILED DESCRIPTION OF THE INVENTION Preparation of Bioconjugate Complexes

As illustrated in FIG. 1, bioconjugate complexes of General Formula I are prepared by a three-step process in which:

(1) A phenylboronic acid complexing reagent of General Formula V, derived from a compound preferably selected from either 4-aminosalicylic acid and 5-aminosalicylic acid, wherein group X is selected from either OH, NH₂, NH-alkyl, NHC₆ H₅, NH-aryl, NHCH₂ -aryl, NHCH₂ CH₂ OH, NHCOCH₂ CH₂ OH, NHOH, NHO-alkyl and NHOCH₂ -aryl wherein alkyl denotes an alkyl hydrocarbon moiety of from 1 to 4 carbons in length, wherein the chains may be branched, wherein aryl is selected from either an aromatic ring, a substituted aromatic ring and fused aromatic rings, and wherein group X is preferably selected from, but not limited to, either OH, NH₂, NHCH₃, NHC₆ H₅, NHCH₂ CH₂ OH, NHCOCH₂ CH₂ OH, NHOH, NHOCH₃, wherein group Y is selected from either O, S, NH, N-alkyl, N-aryl and NCH₂ -aryl, wherein alkyl and aryl are as were previously defined, and wherein group Y is preferably O, wherein group Z comprises a spacer selected from either an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain, up to 16 carbon equivalents in length, wherein either chain may contain intermediate amide and disulfide bonds, and wherein group Z is preferably selected from, but not limited to, either (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to 4, and wherein group R, is preferably selected from, but not limited to, either amino, hydrazide, thiol, isothiocyanate, bromoacetamide, iodoacetamide, maleimide N-hydroxysuccinimidyl ester and N-hydroxysulfosuccinimidyl ester moities, is condensed with a bioactive species to prepare a phenylboronic acid complexing semiconjugate;

(2) A phenylboronic acid reagent of General Formula VII, derived from a compound preferably selected from, but not limited to, either (3-aminophenyl)boronic acid and (4-carboxy-phenyl)boronic acid, wherein group Z* comprises a spacer selected from either an alkyl or a polyethyleneglycol chain, of from 1 to 16 carbon equivalents in length, wherein the chain may contain intermediate amide and disulfide bonds, and wherein group Z* is preferably selected from, but not limited to, either (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to 4, and wherein group R is preferably selected from, but not limited to, either amino, hydrazide, thiol, isothiocyanate, bromoacetamide, iodoacetamide, maleimide N-hydroxysuccinimidyl ester and N-hydroxysulfosuccinimidyl ester moities, is condensed with a bioactive species to prepare a phenylboronic acid semiconjugate;

(3) The phenylboronic acid complexing semiconjugate, prepared as described in (1) above, and the phenylboronic acid semiconjugate, prepared as described in (2) above, are reacted with one another to afford a bioconjugate complex of General Formula I, wherein BAS and BAS* are bioactive species, and wherein groups X, Y, Z and Z* are as were previously described.

As illustrated in FIG. 2, bioconjugate complexes of General Formula II are prepared by a two-step process in which:

(1) A phenylboronic acid reagent of General Formula VII, derived from a compound preferably selected from either (3-aminophenyl)boronic acid and (4-carboxyphenyl)boronic acid, wherein groups Z* and R are as were previously described, is condensed with a bioactive species to prepare phenylboronic acid semiconjugates;

(2) The phenylboronic acid semiconjugates, prepared as described in (1) above, are reacted with a phenylboronic acid complexing cross-linking reagent of General Formula VI, derived from a compound preferably selected from either 4-aminosalicylic acid and 5-aminosalicylic acid, wherein groups X, Y and Z are as were previously described, to afford a bioconjugate complex of General Formula II, wherein groups X, Y, Z, Z* and BAS* are as were previously described.

As illustrated in FIG. 3, bioconjugate complexes of General Formula III are prepared by a two-step process in which:

(1) A phenylboronic acid complexing reagent of General Formula V, wherein groups X, Y and Z are as were previously defined, is condensed with a bioactive species to prepare phenylboronic acid complexing semiconjugates;

(2) The phenylboronic acid complexing semiconjugates, prepared as described in (1) above, are reacted with a phenylboronic acid cross-linking reagent of General Formula VIII, derived from (3-aminophenyl)boronic acid, wherein group Z* is as was previously defined, to afford a bioconjugate complex of General Formula III, wherein groups X, Y, Z, Z* and BAS are as were previously described.

As illustrated in FIG. 4, bioconjugate complexes of General Formula IV are prepared by a two-step process in which:

(1) A phenylboronic acid complexing reagent of General Formula V, wherein groups X, Y and Z are as were previously defined, is condensed with a bioactive species to prepare phenylboronic acid complexing semiconjugates;

(2) The phenylboronic acid complexing semiconjugates, prepared as described in (1) above, are reacted with a phenylboronic acid cross-linking reagent of General Formula IX, derived from (4-carboxyphenyl)boronic acid, wherein group Z* is as was previously defined, to afford a bioconjugate complex of General Formula IV, wherein groups X, Y, Z, Z* and BAS are as were previously described. ##STR6##

Phenylboronic acid complexing reagents of General Formulas V and VI are described in greater detail in my copending application Ser. No. 08/188,531, now U.S. Pat. No. 5,594,157, which is incorporated herein by reference. Reagents of General Formula V are preferably selected from, but not limited to, either 5-iodoacetamidosalicylic acid, 2-hydroxy-4-iodoacetamidobenzohydroxamic acid, N-4-(3-hydrazidopropionamido)-2-hydroxybenzohydroxamic acid and N-hydroxysuccinimidyl 2-hydroxy-N-4-succinamido-O-methylbenzohydroxamic acid. Phenylboronic acid complexing cross-linking reagents of General Formula VI are preferably selected from, but not limited to, either N,N-1,4-bis-succinamido-(2-hydroxybenzohydroxamic acid), N,N-1,6-bis-adiptamido-(2-hydroxybenzohydroxamic acid) and (3,3'-bis-dithiopropionamido)-2-hydroxy-O-methylbenzo-hydroxamic acid.

Synthesis of Phenylboronic Acid Reagents of General Formula VII

The syntheses of phenylboronic acid reagents of General Formula VII (see FIG. 1) are set forth in FIG. 5. Reagents of General Formula VII are derived from a compound preferably selected from, but not limited to, either (3-aminophenyl)boronic acid, (4-carboxyphenyl)boronic acid and N-(6-nitro-3-dihydroxyborylphenyl)succinamic acid.

Reagents of General Formula VII are preferably selected from, but not limited to, either (3-isothiocyanatophenyl)boronic acid (FIG. 5, #1), (5-carboxy-3-isothiocyanatophenyl)boronic acid, (3-iodoacetamidophenyl)boronic acid (FIG. 5, #2), (3-maleimidophenyl)boronic acid (FIG. 5, #3), (3-dihydroxyborylphenyl)succinamic acid succinimidyl ester (FIG. 5, #4) and (3-dihydroxyborylphenyl)succinamic acid hydrazide (FIG. 5, #5). The synthetic preparation of (3-isothiocyanatophenyl)boronic acid and (5-carboxy-3-isothiocyanatophenyl)boronic acid has recently been reported (Linder, K. E., Wen, M. D., Nowotnik, D. P., Malley, M. F., Gougoutas, J. Z., Nunn, A. D. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 160-170, and Linder, K. E., Wen, M. D., Nowotnik, D. P., Ramalingam, K., Sharkey, R. M., Yost, F., Narra, R. K., Nunn, A.D. and Eckelman, W. C. (1991) Bioconjugate Chem., 2, 407-415).

Synthesis of Phenylboronic Acid Reagents of General Formulas VIII & IX ##STR7##

Phenylboronic acid reagents of General Formula VIII are prepared by condensation of (3-aminophenyl)boronic acid with an activated dicarboxylic acid preferably selected from, but not limited to, either succinyl chloride, adipoyl chloride, adiptic acid diisobutylcarbonate, suberoyl chloride, 3,3'-dithiopropionyl chloride and 3,6,9-trioxaundecanedioyl chloride and 3,6,9-trioxa-undecanedioic acid diisobutylcarbonate. The preparation of a single reagent of General Formula VIII has been previously reported (Burnett, T. J., Peebles, H. C. and Hageman, J. H. (1980) Biochem. Biophys. Research Commun., 96, 157-162). ##STR8##

Phenylboronic acid reagents of General Formula IX are prepared by activation of (4-carboxyphenyl)boronic acid with N,N-dicyclohexylcarbodiimide, followed by condensation with a diamine, preferably selected from, but not limited to, either 1,4-butanediamine, 1,6-hexanediamine and 2,2'-dithiodiaminoethane (H₂ NCH₂ CH₂ SSCH₂ CH₂ NH₂).

Bioconjugate Complexes

Bioconjugate complexes of General Formulas I to IV are prepared in buffered aqueous solutions preferably selected from, but not limited to, acetate, citrate, phosphate and carbonate buffers. Borate and Tris buffers should be avoided due to their potential for complexation with phenylboronic acid complexing moities and phenylboronic acid moieties, respectively. The bioconjugate complex is formed within 1 to 15 minutes at room temperature. The reaction is insensitive to variations in ionic strength over the range 0.01 to 2 molar. Stability of the complex increases with increasing temperature, limited only by the volatility of the buffer. Addition of organic solvents including acetonitrile, methanol, ethanol, isopropanol, butanol, N,N-dimethylformamide and dimethylsulfoxide serves to further stabilize bioconjugates. Chaotropic reagents (protein denaturants) including urea, guanidine hydrochloride and formamide also serve to further stabilize bioconjugates, if and when the bioactive species is tolerant of their presence. Bioconjugate complexes may be purified by desalting, dialysis, size exclusion chromatograpihy and electrophoresis. Bioconjugate complexes are stable upon removal of water, and can be lyophilized for storage.

Ionization of phenylboronic acid is an important factor in bioconjugate complex formation, in that, upon ionization boron changes from trigonal coordination (having average bond angles of 120° and average bond lengths of 1.37 Å) to the tetrahedrally coordinated anion (having average bond angles of 109° and average bond lengths of 1.48 Å). FIG. 6 illustrates both the trigonal and tetrahedral forms of bioconjugate complexes of General Formula I (wherein groups Z, Z*, BAS and BAS* have been omitted for clarity).

Phenylboronic acids vary in pK_(a) between approximately 5.2 and 9.2. Bioconjugate complexes of General Formulas I, II and IV, derived from (4-carboxyphenyl)boronic acid, have approximate pK_(a) values in the range 6.5 to 7.5. Bioconjugate complexes of General Formulas I, II and III, derived from (3-aminophenyl)boronic acid, have approximate pK_(a) values in the range 8.0 to 9.0. Complexes of General Formulas I to IV derived, from either (3-amino-2-nitrophenyl)boronic acid, (3-amino-5-nitrophenyl)boronic acid, and (3-amino-6-nitrophenyl)boronic acid have intermediate pK_(a) values.

As a general rule, the pK_(a) of a complex of General Formulas I to IV is approximately one pH unit below that of the phenylboronic acid from which the complex was prepared.

For each phenylboronic acid moiety, the stability of the bioconjugate complex, at a given pH, is determined by substituent groups X and Y. For semiconjugates derived from reagents of General Formulas V and VI, individuals skilled in the art will recognize that a number of tautomeric and conformation isomers exist in equilibrium, as illustrated in FIG. 7 (BAS omitted for clarity). Each of the isomers depicted in FIG. 7 may contribute to the phenylboronic acid complex, as illustrated in FIG. 8 (BAS, Z* and BAS* omitted for clarity).

Bioconjugate complexes of General Formulas I to IV, wherein both groups X and Y are preferably O, and wherein the phenylboronic acid moiety is derived from (3-aminophenyl)boronic acid, are stable in acidic aqueous solutions of approximate pH range 2.5 to 4.5. This range of pH stability results from the requirement that the substituent group X associated with the phenylboronic acid complexing moiety from which the bioconjugate complex was derived must posses an acidic hydrogen if a stable complex is to result. As illustrated in FIG. 9, bioconjugate complexes will only form at low pH where the salicylic acid moiety is protonated. Nevertheless, below pH 2.5 the complex is unstable, due to acid catalyzed hydrolysis.

Bioconjugate complexes of General Formulas I to IV, wherein group X is preferably selected from either NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH and NCOCH₂ CH₂ OH, wherein group Y is preferably selected from either O and NH, and wherein the phenylboronic acid moiety is derived from (3-aminophenyl)boronic acid, are stable in buffered alkaline aqueous solutions over the approximate pH range 8.5 to 11.5. This range of pH stability results from the requirement that only the phenylboronate anion affords a stable complex. Nevertheless, above pH 11.5 the complex is unstable, due to base catalyzed hydrolysis. Bioconjugate complexes which exhibit stability only under either acidic or alkaline conditions are useful for reversible conjugation, whereby the bioconjugate complex may be disassociated by appropriate adjustment of the pH.

Bioconjugate complexes of General Formulas I, II and III, wherein group X is preferably selected from either NOH and NOCH₃, wherein group Y is preferably O, and wherein the phenylboronic acid moiety is derived from (3-aminophenyl)boronic acid, constitute a special case in which complexes are stable in buffered aqueous solutions over the broad approximate pH range 2.5 to 11.5. This wide range of pH stability is thought to result from the presence of a coplanar 1,3-diol complexing moiety associated with the enol form of the 2-hydroxybenzohydroxamic acid moiety. Alternatively, pH stability may result from the low effective pKa of the phenylboronic acid in the complex which results from reaction of a hydroxamic acid anion (CON⁻ OH) with phenylboronic acid forming a dative bond which fills the outer electron shell of the boron atom. Bioconjugate complexes of this type form in an essentially irreversible manner, as they may only be disassociated by adjustment of the pH to above 11.5, or below 2.5, or by competitive dissociation with borate buffer.

Characterization of Semiconjugates

Semiconjugates prepared as illustrated in FIG. 1, wherein the bioactive species is a protein, may be characterized with respect to the number of phenylboronic acid complexing moieties or phenylboronic acid moities incorporated (degree of substitution) per protein molecule. Semiconjugates having pendant phenylboronic acid complexing moieties may be characterized by reaction with an excess of a fluorescent phenylboronic acid reagent, in a buffered aqueous solution of appropriate pH, to afford a bioconjugate complex of General Formula X, wherein X, Y, Z, Z* and BAS are as were previously defined, and wherein a fluorescent moiety F* is the bioactive species. The fluorescent moiety F* is preferably selected from, but not limited to, fluorescein, rhodamine X, tetramethylrhodamine, Texas Red, phycoerythrin and allophycocyanin. After removal of the excess reagent by desalting, dialysis or size exclusion chromatography, the bioconjugate complex of General Formula X is subjected to spectroscopic analysis, and the number of phenylboronic acid complexing moieties calculated by comparing the ratio of the absorption at 280 nm, which denotes the total protein concentration, to the absorption at a wavelength characteristic of the fluorophore (λ_(max)). The preparation of a single fluorescent phenylboronic acid has been previously reported (Burnett, T. J., Peebles, H. C. and Hageman, J. H. (1980) Biochem. Biophys. Research Commun., 96, 157-162). ##STR9##

Similarly, semiconjugates having pendant phenylboronic acid moieties may be characterized by reaction with an excess of a fluorescent phenylboronic acid complexing reagent, in a buffered aqueous solution of appropriate pH, to afford a bioconjugate complex of General Formula XI, wherein X, Y, Z, Z* and BAS* are as were previously defined, and wherein a fluorescent moiety F is the bioactive species. The fluorescent moiety F is preferably selected from, but not limited to, fluorescein, rohdamine X, tetramethylrhodamine, Texas Red, phycoerythrin and allophycocyanin. After removal of the excess reagent by desalting, dialysis or size exclusion chromatography, the bioconjugate complex of General Formula XI is subjected to spectroscopic analysis, and the number of phenylboronic acid moieties calculated by comparing the ratio of the absorption at 280 nm, which denotes the total protein concentration, to the absorption at a wavelength characteristic of the fluorophore (λ_(max)). Semiconjugates derived from other high molecular weight bioactive species which are suitable for purification by desalting, dialysis or size exclusion chromatography may be characterized in an analagous manner.

EXAMPLE I Preparation of an Amine Reactive Reagent of General Formula VII N-(3-Dihydroxyborylphenyl)succinamic Acid, Succinimidyl Ester

Succinic anhydride (5.00 grams, 0.05 mole) and (3-aminophenyl)boronic acid (7.75 grams, 0.05 mole) are dissolved in anhydrous pyridine (40 ml), and then allowed to stand overnight at room temperature. Water (20 ml) is added and the resulting solution allowed to stand for 1 hour. The product is then concentrated on a rotary evaporator at 85°-90° C. The resulting aqueous solution is frozen in a dry-ice-acetone-slurry and lyophilized overnight. The lyophilized product is dissolved in water (50 ml) and acidified with concentrated HCl to approximately pH 1.0. The acidified solution is cooled in an ice bath for 1 hour, and the precipitate collected by filtration. The precipitate is recrystallized from boiling water (200 ml) and dried overnight in vacuo over NaOH pellets, to afford 8.60 grams (70% yield) of N-(3-dihydroxyborylphenyl)succinamic acid. Homogeneous by TLC (CHCl₃ /CH₃ OH/CH₃ COOH; 60:35:5), R_(f) =0.5. Melting point 186°-188° C. The structure was confirmed by 300 MHz, ¹ H NMR, spectrometry in d₆ -DMSO.

N-(3-dihydroxyborylphenyl)succinamic acid (16.0 grams, 0.063 mole) is dissolved in dry DMF (80 ml). To the solution is added N,N-dicyclohexylcarbodiimide (14.3 grams, 0.069 mole) followed by N-hydroxysuccinimide (8.05 grams, 0.070 mole). The reaction is stirred overnight at room temperature. N,N-dicyclohexylurea is filtered from the solution, and the filtrate extracted with ethyl acetate (200 ml). The extract is washed with water (3×400 ml) and saturated NaCl (400 ml). The water wash was back-extracted with ethyl acetate (200 ml), the extracts combined, dried over anhydrous Na₂ SO₄, and concentrated on a rotary evaporator to afford 12.5 grams (57% yield) of N-(3-dihydroxyboryl-phenyl)succinamic acid, succinimidyl ester. Purity estimated at 98% by TLC (CHCl₃ /CH₃ OH/CH₃ COOH; 85:10:5), R_(f) =0.7. The structure was confirmed by 300 MHz, ¹ H NMR spectrometry in d₆ -DMSO.

EXAMPLE II Applications of an Amine Reactive Reagent of General Formula VII

Proteins may be modified with amine reactive phenylboronic acid reagents of General Formula VII by reaction with the side-chain ε-amino groups of lysine residues, to afford semiconjugates having pendant phenylboronic acid moieties covalently affixed to the protein through stable amide bonds. N,N-Dimethylformamide and dimethylsulfoxide are the solvents of choice. Mildly alkaline aqueous buffers, in the pH range 7.8 to 8.8, and preferably 100 mM bicarbonate buffer, pH 8.2, should be employed so as to insure that the amino group is unprotonated, while minimizing hydrolysis of the N-hydroxysuccinimidyl ester. Activated N-hydroxysuccimidyl esters have been observed to interact with phenylboronic acids in alkaline aqueous solutions resulting in a significant reduction in their reactivity. To overcome this limitation, aqueous reactions involving N-(3-dihydroxyborylphenyl)succinamic acid, succinimidyl ester should only be undertaken in the presence of at least a 10-fold molar exess of a phenylboronic acid complexing ligand. Compounds found to be useful in this regard include mannitol and catechol. Complexes temporarily prepared for this purpose may be readily dissociated upon neutralization of the solution. Primary amine containing buffers including Tris and glycine must be avioded, due to their potential reactivity. Solid-phase supports having pendant primary amine moieties, including blotting membranes and microtiter plates, may be functionalized by reaction with phenylboronic acid reagents of General Formula VII to afford solid-phase supports having pendant phenylboronic acid moieties.

EXAMPLE III Preparation of a Thiol Reactive Reagent of General Formula VII (3-Maleimidophenyl)boronic Acid

Ethyl acetate (400 ml) is cooled in an ice bath to approximately 0° C. Maleimide (7.76 grams) is added to the cooled solvent with strring, followed by N-ethylmorpholine (10.19 ml). Methyl chloroformate (6.26 ml) is added dropwise from an addition funnel at an appropriate rate so as to maintain the temperature of the reaction below 3° C. After completion of the addition, the reaction is stirred for an additional 30 min. while maintaining the temperature below 3° C. The resulting mixture is filtered through a Buchner funnel, and the precipitate washed with a small volume of ethyl acetate. The filtrate and wash are combined, and then washed with ice cold water (100 ml). The organic phase is dried over anhydrous Na₂ SO₄, and then concentrated on a rotary evaporator. The product is dissolved in a mixture of ethyl acetate and isopropyl ether (40:60 v/v, 75 ml) in a water bath at 60° C., and then allowed to recrystallize at room temperature. Crystals of N-methoxycarbonylmaleimide are washed with isopropyl ether (2×20 ml), and then dried overnight in vacuo.

(3-Aminophenyl)boronic acid (1.26 grams, 0.01 mole) is dissolved in saturated NaHCO₃ (50 ml) by briefly heating the mixture on a hot plate. The solution is cooled in an ice bath to approximately 0° C., and N-methoxycarbonylmaleimide (1.55 grams, 0.01 mole) added with vigorous stirring. After 10 min. the solution is diluted with water (200 ml) and then stirred at room temperature for 30 to 40 min. The pH is adjusted to approximately 5.5 by addition of 1M H₂ SO₄, and the precipitate collected by filtration. The precipitate is washed with 1M H₂ SO₄ (2×50 ml), and then dried overnight in vacuo over NaOH pellets to afford 1.39 grams (64% yield) of (3-maleimidophenyl)boronic acid. The structure was confirmed by 300 MHz, ¹ H NMR spectrometry in d₆ -DMSO.

EXAMPLE IV Applications of a Thiol Reactive Reagent of General Formula VII

Proteins containing disulfide bonds (cystine residues) or cysteine residues may be modified with thiol reactive phenylboronic acid reagents of General Formula VII. Disulfide bonds are first reduced, if required, by reaction with 2-mercaptoethanol or dithiothreitol, in an alkaline aqueous buffer. The excess reducing reagent is removed by dialysis or desalting, and the protein reacted with (3-maleimidophenyl)boronic acid in 25 to 100 mM phosphate buffer, pH 7.0 to 7.5, for 1 hour at 4° C., to afford a semiconjugate having pendant phenylboronic acid moieties covalently affixed to the protein. Proteins which lack thiol moieties may be functionalized by reaction with a thiolating reagent, and then modified as described above. Thiolating reagents which have proven useful for this purpose include N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate, N-hydroxy-succinimidyl S-acetylthioacetate and 2-iminothiolane.

EXAMPLE V Preparation of an Aldehyde Reactive Reagent of General Formula VII N-(3-Dihydroxyborylphenyl)succinamic Acid Hydrazide

Methanol (10 ml) is cooled in an ice bath to approximately 0° C., and thionyl chloride (1 ml) slowly added. To the resulting stirred solution is added N-(3-dihydroxyborylphenyl)-succinamic acid (1.25 grams, 0.005 mole), prepared as described in Example I, and the reaction is stirred overnight at room temperature. The solution is concentrated on a rotary evaporator to afford a white crystalline material which is coevaporated twice from methanol (2×10 ml) to remove residual thionyl chloride. The product is dissolved in methanol (5 ml) and hydrazine hydrate (1 ml) added. The resulting solution is stirred overnight at room temperature. A precipitate forms within a few hours. The precipitate is collected by filtration, washed with cold methanol and dried overnight in vacuo over NaOH pellets to afford 1.11 grams (88% yield) of N-(3-dihydroxyborylphenyl)succinamic acid hydrazide. The structure was confirmed by 300 MHz, ¹ H NMR spectrometry in d₆ -DMSO.

EXAMPLE VI Application of an Aldehyde Reactive Reagent of General Formula VII

Glycoproteins, and particularly antibodies, may be conjugated with an aldehyde reactive phenylboronic acid hydrazide reagent after treatment of the protein with from 5 to 20 mM sodium meta periodate (NaIO₄), in from 0.1 to 0.5M sodium acetate buffer at pH 5 to 6, containing up to 0.2M sodium chloride, at 0° C., for from 30 minutes to 4 hours. The excess periodate is removed by desalting, and the activated protein, having pendant adjacent aldehyde moieties resulting from periodate oxidation of carbohydrate residues having 1,2-diol moieties, is condensed with the hydrazide reagent, for from 1 to 24 hours at room temperature, to afford a semiconjugate having pendant phenylboronic acid moities covalently appended to the protein through a Schiff base (animine) type linkage. The stability of the linkage to the protein may be increased, if desired, by mild sodium cyanoborohydride reduction of the Schiff base to the corresponding alkylamine.

It is important to note that periodate oxidation of a glycoprotein activates the protein toward reaction with a hydrazide type reagent while simultaneously removing most naturally occuring phenylboronic acid complexing moities (coaxial 1,2-diols) associated with glycoproteins. 

I claim:
 1. A bioconjugate complex having the general formula: ##STR10## wherein group X is selected from the group consisting of O, NH, N-alkyl, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH, and NO-alkyl, wherein alkyl denotes alkyl hydrocarbon moiety of from 1 to 4 carbons in length and which may be linear or branched,wherein group Y is selected from the group consisting of O, S, NH, and N-alkyl, wherein groups Z and Z* comprise a spacer selected from an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain up to 16 carbon equivalents in length, wherein either chain may contain intermediate amide and disulfide bonds, and wherein group BAS* is a bioactive species.
 2. The bioconjugate complex of claim 1 wherein group X is selected from the group consisting of O, NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH and NOCH₃, wherein group Y is O, and wherein groups Z and Z* are selected from the group consisting of (CH₂)_(n), wherein n=1 to 4, and (CH₂ CH₂ O)_(n'), wherein n'=2 to
 4. 3. The bioconjugate complex of claim 1 wherein said bioactive species is selected from the group consisting of proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes, cells, drugs, radionuclides, toxins, haptens, inhibitors, fluorophores, ligands and solid-phase supports.
 4. A bioconjugate complex having the general formula: ##STR11## wherein group X is selected from the group consisting of O, NH, N-alkyl, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH, and NO-alkyl, wherein alkyl denotes an alkyl hydrocarbon moiety of from 1 to 4 carbons in length and which may be linear or branched,wherein group Y is selected from the group consisting of O, S, NH, and N-alkyl, wherein groups Z and Z* comprise a spacer selected from an alkyl of from 1 to 16 carbon equivalents in length and a polyethyleneglycol chain up to 16 carbon equivalents in length, wherein the chain may contain intermediate amide and disulfide bonds, and wherein group BAS is a bioactive species.
 5. The bioconjugate complex of claim 4 wherein group X is selected from the group consisting of O, NH, NCH₃, NC₆ H₅, NCH₂ CH₂ OH, NCOCH₂ CH₂ OH, NOH and NOCH₃, wherein group Y is O, and wherein groups Z and Z* are selected from the group consisting of (CH₂)_(n), wherein n=1 to 5, and (CH₂ CH₂ O)_(n'), wherein n'=2 to
 4. 6. The bioconjugate complex of claim 4 wherein said bioactive species is selected from the group consisting of proteins, peptides, polysaccharides, hormones, nucleic acids, liposomes, cells, drugs, radionuclides, toxins, haptens, inhibitors, fluorophores, ligands and solid-phase supports. 